Description: The oxadiazole antibacterials target the bacterial cell wall and are bactericidal. We investigated the synergism of ND-421 with the commonly used β-lactams and non-β-lactam antibiotics by the checkerboard method and by time-kill assays. ND-421 synergizes well with β-lactam antibiotics, and it also exhibits a long postantibiotic effect (4.7 h). We also evaluated the in vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant infection model (1.49 log 10 bacterial reduction for ND-321 versus 0.36 log 10 for linezolid with NRS119) and acts synergistically with β-lactam antibiotics in vitro and in vivo , and the combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log 10 bacterial reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain would have been resistant to oxacillin otherwise.

Description: Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt , was cloned from the strain, and three other genes, metF , dhc , and purU , which are involved in THF metabolism, were found to be located downstream of dmt . A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. IMPORTANCE Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.

Description: The degradation of the herbicide dicamba is initiated by demethylation to form 3,6-dichlorosalicylate (3,6-DCSA) in Rhizorhabdus dicambivorans Ndbn-20. In the present study, a 3,6-DCSA degradation-deficient mutant, Ndbn-20m, was screened. A cluster, dsmR1DABCEFGR2 , was lost in this mutant. The cluster consisted of nine genes, all of which were apparently induced by 3,6-DCSA. DsmA shared 30 to 36% identity with the monooxygenase components of reported three-component cytochrome P450 systems and formed a monophyletic branch in the phylogenetic tree. DsmB and DsmC were most closely related to the reported [2Fe-2S] ferredoxin and ferredoxin reductase, respectively. The disruption of dsmA in strain Ndbn-20 resulted in inactive 3,6-DCSA degradation. When dsmABC , but not dsmA alone, was introduced into mutant Ndbn-20m and Sphingobium quisquiliarum DC-2 (which is unable to degrade salicylate and its derivatives), they acquired the ability to hydroxylate 3,6-DCSA. Single-crystal X-ray diffraction demonstrated that the DsmABC-catalyzed hydroxylation occurred at the C-5 position of 3,6-DCSA, generating 3,6-dichlorogentisate (3,6-DCGA). In addition, DsmD shared 51% identity with GtdA (a gentisate and 3,6-DCGA 1,2-dioxygenase) from Sphingomonas sp. strain RW5. However, unlike GtdA, the purified DsmD catalyzed the cleavage of gentisate and 3-chlorogentisate but not 6-chlorogentisate or 3,6-DCGA in vitro . Based on the bioinformatic analysis and gene function studies, a possible catabolic pathway of dicamba in R. dicambivorans Ndbn-20 was proposed. IMPORTANCE Dicamba is widely used to control a variety of broadleaf weeds and is a promising target herbicide for the engineering of herbicide-resistant crops. The catabolism of dicamba has thus received increasing attention. Bacteria mineralize dicamba initially via demethylation, generating 3,6-dichlorosalicylate. However, the catabolism of 3,6-dichlorosalicylate remains unknown. In this study, we cloned a gene cluster, dsmR1DABCEFGR2 , involved in 3,6-dichlorosalicylate degradation from R. dicambivorans Ndbn-20, demonstrated that the cytochrome P450 monooxygenase system DsmABC was responsible for the 5-hydroxylation of 3,6-dichlorosalicylate, and proposed a dicamba catabolic pathway. This study provides a basis to elucidate the catabolism of dicamba and has benefits for the ecotoxicological study of dicamba. Furthermore, the hydroxylation of salicylate has been previously reported to be catalyzed by single-component flavoprotein or three-component Rieske non-heme iron oxygenase, whereas DsmABC was the only cytochrome P450 monooxygenase system hydroxylating salicylate and its methyl- or chloro-substituted derivatives.

Description: Brain heart infusion agar containing 3 mg/liter vancomycin (BHI-V3) was used to screen for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). There was markedly greater biofilm formation by isolates that grew on BHI-V3 than by strains that did not grow on BHI-V3. Increased biofilm formation by hVISA may be mediated by FnbA- and polysaccharide intercellular adhesin-dependent pathways, and upregulation of atlA and sarA may also contribute to enhanced biofilm formation by hVISA upon prolonged exposure to vancomycin.